title: Expression in Haloferax volcanii of 3-hydroxy-3-methylglutaryl coenzyme A synthase facilitates isolation and characterization of the active form of a key enzyme required for polyisoprenoid cell membrane biosynthesis in halophilic archaea.
abstract: Enzymes of the isoprenoid biosynthetic pathway in halophilic archaea remain poorly characterized, and parts of the pathway remain cryptic. This situation may be explained, in part, by the difficulty of expressing active, functional recombinant forms of these enzymes. The use of newly available expression plasmids and hosts has allowed the expression and isolation of catalytically active Haloferax volcanii 3-hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (EC 2.3.310). This accomplishment has permitted studies that represent, to the best of our knowledge, the first characterization of an archaeal hydroxymethylglutaryl CoA synthase. Kinetic characterization indicates that, under optimal assay conditions, which include 4 M KCl, the enzyme exhibits catalytic efficiency and substrate saturation at metabolite levels comparable to those reported for the enzyme from nonhalophilic organisms. This enzyme is unique in that it is the first hydroxymethylglutaryl CoA synthase that is insensitive to feedback substrate inhibition by acetoacetyl-CoA. The enzyme supports reaction catalysis in the presence of various organic solvents. Haloferax 3-hydroxy-3-methylglutaryl CoA synthase is sensitive to inactivation by hymeglusin, a specific inhibitor known to affect prokaryotic and eukaryotic forms of the enzyme, with experimentally determined Ki and kinact values of 570 ± 120 nM and 17 ± 3 min(-1), respectively. In in vivo experiments, hymeglusin blocks the propagation of H. volcanii cells, indicating the critical role that the mevalonate pathway plays in isoprenoid biosynthesis by these archaea.
authors.affiliation: Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City, Kansas City, Missouri, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: VanNice
authors.forename: D Andrew
authors.initials: DA
authors.lastname: Skaff
authors.forename: Gerald J
authors.initials: GJ
authors.lastname: Wyckoff
authors.forename: Henry M
authors.initials: HM
authors.lastname: Miziorko
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism; D015870:Gene Expression; D019848:Gene Expression Regulation, Archaeal; D015971:Gene Expression Regulation, Enzymologic; D019622:Haloferax volcanii / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016415:Sequence Alignment; D013729:Terpenes / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D004791:Enzyme Inhibitors; D013729:Terpenes; C010667:acetoacetyl CoA; D006904:Hydroxymethylglutaryl-CoA Synthase